Picornaviruses are positive-strand RNA viruses that contain a long open-reading-frame encoding a poly-protein. Cardioviruses and aphthoviruses are two genera of picornaviruses. The 5'-end non-coding sequences for these genera are typically 750 to 1,300 nucleotides in length. Some strains of cardioviruses and aphthoviruses have a homopolymeric non-coding poly(C) tract which is located about 150 to 330 bases from the 5'-end of the RNA strand.
The length of the poly(C) tract in cardioviruses and aphthoviruses is usually between 60 to 200 bases and the tract may include discontinuities, such as the insertion of a U residue within the stretch of poly(C). With respect to wild-type virus, by poly(C) tract, we mean any stretch of residues longer than 20 residues which is at least 75% C, ends and begins with a sequence of at least four consecutive C residues, and is within the 5' non-coding region of a wild-type picornavirus genome. Our convention is to count the discontinuities within the tract when we refer to the length of the tract. For example, a tract of C.sub.13 UC.sub.10 is a tract of 24 residues.
Both the length of the poly(C) tract and the particular discontinuities are characteristics of a particular strain of cardiovirus or aphthovirus. Examples of poly(C) tractcontaining cardioviruses are Mengoviruses, EMCV (encephalomyocarditis virus), ME (Maus Elberfeld), Columbia SK, and MM. Foot and mouth disease virus (FMDV) is an example of an aphthovirus containing a poly(C) tract.
Mengovirus and EMCV had been considered primarily murine (rodent) in their host range. However, isolated reports of cardiovirus infections in non-murine mammals (e.g., humans, non-human primates, swine, elephants, and lions) have been published. Very recently, improved serotypic detection methods have demonstrated the extent and prevalence of cardiovirus-induced infections among animals. For example, the "mystery disease" affecting pig herds in Indiana, Iowa, Minnesota and other states is now attributed to EMCV. Joo, et al., Arch. Virol. 100: 131-134 (1989). (The disclosures of all articles recited herein are incorporated by reference as if fully set forth below.) Recently, some captive primate colonies, such as domestic breeding facilities and zoos, have reported loss of animals because of EMCV infections. The clinical signs of EMCV infection are high rate of stillbirth, fever, lack of appetite, and late-term abortions. Baby pigs affected with EMCV are weak and have labored breathing. Autopsies of infected animals have revealed enlarged hearts with white striations or spots.
The host range of FMDV is primarily bovine. FMDV infection is believed to cause a significant loss of cattle in European and certain other countries.
There is therefore a great need for vaccines against disease caused by EMCV, FMDV, and other picornaviruses. Certain currently available vaccines, such as an EMCV vaccine from Oxford Veterinary Laboratories, Inc. (Worthington, Minn.), are "killed virus" vaccines. A killed virus vaccine contains a wild-type virus that has been inactivated, usually through chemical means. These vaccines have the disadvantage of shorter duration of immunity than an attenuated live virus vaccine. (An "attenuated" virus is a live virus that has a lessened capability to cause disease when compared to the wild-type virus.)
Recent inoculation studies in our laboratory with mice have demonstrated that a genetically engineered Mengovirus containing an artificially shortened poly(C) tract was attenuated compared to wild-type viruses. Osorio, et al. "Attenuation of Mengovirus Pathogenicity through Recombinant Engineering of the Poly(C) Tract," in Europic 89, Sixth Meeting of the European Study Group on the Molecular Biology of Picornaviruses, Bruges, Belgium, Sep. 10-16, 1989 (Abstract).
Although use of short poly(C) tract Mengovirus has been reported with respect to mice, picornavirus vaccines effective in pigs, monkeys and other animals are still needed. Living things are classified according to the following scheme: Kingdom, Phylum, Class, Order, Family, Genus, and Species. While all mammals belong to the same class, primates (e.g. monkeys and humans), rodents, and pigs each belong to different orders. Prior to our invention, there were no known attenuated vaccines that were reactive across different taxonomic orders.